Golgi and Secretion

Organelles of the cell

pictured

Lysosome

An organelle in the cytoplasm of eukaryotic cells containing degradative enzymes enclosed in a membrane. Proteins that are destined to go to the cell surface to be secreted, to go to lysosomes, are going via the ER-Golgi system.

mannose-6-phosphate

Another function of the golgi is mannose-6-phosphate. when it's in the golgi, you have 2 enzymes that will ultimately result in the phosphorylation of the mannose. Not a typically kinase phosphorylation reaction, that we will talk about in signaling. It is where you have a two-step process and the net result is mannose-6-phosphate. If I did the numbering system of mannose [see right]. It is the 6th carbon of the mannose that has the phosphate group put on it. In the trans golgi you have mannose-6-phosphate receptors that will cluster into vesicles that are forming with this clathrin coat on it. Dr. Etlinger talked about clathrin coat in receptor-mediated endocytosis. Here is a second place you have this coat - in vesicles targeted for the lysosomes. They go in and hydrolase pops off, then the receptors get recycled back for further use. What causes the pop off? The acidity of the compartment. What causes binding too receptor? Increase in pH of the compartment. The affinity of the receptor for the mannose-6-phosphate is tightly regulated by pH of the lumen. As we go from ER to lysosome, start with neutral and get more acidic. That's very important for biological properties and targeting and movement. Another image from the ER. Mannose-6-phosphate binds to receptor and the receptor goes to lysosome, bringing the hydrolase. This is the mechanism of bringing hydrolases to the lysosome, not the mechanism for bringing proteins to the lysosome for degradation. Where do the bulk proteins that are being degraded come from? Outside the cell or plasma membrane. It is the endocytic vesicles that are bringing in proteins that fuses with endolysosome. Hydrolases are brought in by this pathway.

Golgi apparatus

Golgi apparatus, also called Golgi complex or Golgi body, membrane-bound organelle of eukaryotic cells (cells with clearly defined nuclei) that is made up of a series of flattened, stacked pouches called cisternae. The Golgi apparatus is responsible for transporting, modifying, and packaging proteins and lipids into vesicles for delivery to targeted destinations. It is located in the cytoplasm next to the endoplasmic reticulum and near the cell nucleus. The properties of the golgi are that it is a distillation apparatus. By default, what you can have are proteins that are moving from the ER going into the golgi for further processing. In some cases, there is a retrieval mechanism because a protein needs to reside in the ER for function, and so even if it escapes, there is a way of bringing it back into the ER. Recognize the golgi are basically polarized. The cis face looks more like the ER and the trans face looks more like the plasma membrane, because you are basically going in a vectorial direction and you're getting a distillation. There are different reactions happening in different cisternae and this is what drives in a forward direction (cis stains differently than trans). Functions: Now for the functions. Sulfation is a function. Dr. Newman mentioned you have a lot of ECM proteins, proteoglycans are highly sulfated. Sulfation occurs in golgi, with the proteins made in ER. Proteolysis events: This process is a continuum of the ER, golgi and granules and exocytosis. Proteolysis events are really important and important for diagnostics. Turns out that proteins made in the ER need to be clipped, not totally degraded, to get an active fragment. We have proteins like insulin. Insulin is made in rough ER and looks like (red-green-blue color image). It is one polypeptide chain and in the ER you have the disulfide bridges put on (in black). Insulin when it is secreted looks like (second pic). Insulin has 2 chains - a and b chain. In its precursor form, it is one big unit. So somewhere, this red connecting peptide is cleaved.

EM of mitochondria and golgi

Just by looking at this [mitochondria], you should be able to identify this as the mitochondria based on the cristae within. We will be focusing on [the circled structure], which is the golgi apparatus. As you saw from the pre-lecture video, it is a series of flattened cisternae that are kind of cupped within each other. From that video, you know, you have that cis end [to the right on the golgi] and the internal cupping [to the left] as the trans, or trans golgi network (TGN). You should be able to look at EMs and see rough ER, smooth ER, Golgi and mitochondria.

KDEL sequence

KDEL is a target peptide sequence in the amino acid structure of a protein which prevents the protein from being secreted from the endoplasmic reticulum (ER). A protein with a functional KDEL motif will be retrieved from the Golgi apparatus by retrograde transport to the ER lumen. It also targets proteins from other locations (such as the cytoplasm) to the ER. Proteins can only leave the ER after this sequence has been cleaved off. KDEL stands for individual amino acids - lysine, aspartic acid, glutamic acid, leucine. If you see this anywhere, it will be given as KDEL. KDEL is a motif/patch on the protein that says "I need to go back to the ER".

Mitochondria

Mitochondria are rod-shaped organelles that can be considered the power generators of the cell, converting oxygen and nutrients into adenosine triphosphate (ATP). ATP is the chemical energy "currency" of the cell that powers the cell's metabolic activities.

O-linked glycosylation

Modification of serine [left] and threonine residues. O-linked modification is modifying the oxygen group with sugar moieties. Whereas in N-linked, you have the sugars being added in bulk as one group, in O-linked, you have each sugar added individually. The enzymes that allow for this do not exist in the ER; they exist in the golgi and in the cytoplasm. Where do you expect to find O-linked glycosylated proteins? Inside the nucleus, mitochondria, lysosomes, cytoplasm, golgi, everywhere. Found everywhere because the enzymes are found in both the cytosol and the golgi.

N-linked glycosylation

N-linked glycosylation means sugars put on asparagine residue - it's R group has a free -NH2 on it. It is the nitrogen that is getting glycosylated so it is called N-linked glycosylation. What happens is, if a protein is destined to be N-linked glycosylated, a high mannose sugar group is put on it in the lumen of the rough ER. The enzymes are there. You end up putting a high mannose sugar group on this asparagine then it goes into the golgi where it is tailored so some sugars come off and others go on and you get the mature N-linked glycosylated proteins. [The image on the left] is an integral membrane protein. How do you know which side is the outside? Because the N-linked sugar moieties are always facing outside. Disulfide bridges and GPI linkage both occur in the lumen of the ER, so these modifications bring these modifications out. If you look at the N-linked sugar moiety [right], the blue represents mannose. It immediately tells you that mannose-6-phosphate, you think of N-linked glycosylation. Here is your ribosome [R], here is your signal sequence [green portion in translocon]. This protein is getting translated into the lumen of the ER. The ASN residue is having the high mannose sugar moiety added to it because it is being N-linked glycosylated. We won't get into the mechanism of how the N-linked sugar groups are being formed. Here is the transfer of the precursor molecule onto the ASN (aspargine). Now you have an N-linked glycosylated protein with a lot of mannoses (blue). What is on the plasma membrane is different, so it gets remodeled in the golgi. You have some of these mannoses coming off and other sugar groups being put on, so you have a mature protein. So in the lumen of the golgi you get this remodeling. Where do you expect to find N-linked glycosylations? Secreted, facing outside the plasma membrane, rough ER, lumen of the golgi, and lysosomes. These are restricted to that pathways.

Fibronectin

Protein found in solid form in ECM and soluble form in blood. Important for clotting.

ER Golgi System

Proteins that are destined to go to the cell surface to be secreted, to go to lysosomes, are going via the ER-Golgi system. The ER, Golgi apparatus, and lysosomes are all members of a network of membranes, but they are not continuous with one another. Therefore, the membrane lipids and proteins that are synthesized in the ER must be transported through the network to their final destination in membrane-bound vesicles. Cargo-bearing vesicles pinch off of one set of membranes and travel along microtubule tracks to the next set of membranes, where they fuse with these structures. Trafficking occurs in both directions; the forward direction takes vesicles from the site of synthesis to the Golgi apparatus and next to a cell's lysosomes or plasma membrane. Vesicles that have released their cargo return via the reverse direction. The proteins that are synthesized in the ER have, as part of their amino acid sequence, a signal that directs them where to go, much like an address directs a letter to its destination. Note: Isoprenylated proteins are not released to the outside (When we talk about processes like isoprenylation, these are proteins produced on free ribosomes and are modified on post-translational modification in the cytoplasm and then reside on some internal membranous structure, like the inner leaflet of the plasma membrane, or nuclear lamins, or isoprenylated in the nuclear membrane, etc.)

Proteins destined for cell surface

Proteins that are destined to the cell surface or lysosome are secreted out are going via the ER, into the CGN, the cis cisternae, the medial cisternae, the trans cisternae, the TGN and are trafficked to the right location. But recognize that proteins will not skip cisternae or vesicles do not drive it by skipping cisternae. They will go in a vectorial direction, in the anterograde fashion from the cis to the trans and out. Point mutations can cause proteins to get stuck in ER or golgi and cause disease. You will hear about a point mutation somewhere in the gene product of this protein prevents correct folding. What happens is you get a decreased amount of vasopressin released, and that leads to diabetes insipidus, which you will hear more about in physiology. This is occurring at the level of the ER-Golgi interactions.

Ribosome

Ribosomes are the protein builders or the protein synthesizers of the cell. They are like construction guys who connect one amino acid at a time and build long chains. Ribosomes are special because they are found in both prokaryotes and eukaryotes.

ER Golgi pathway

The anterograde is going from the ER to the cis, medial, trans, and out. Retrograde is going back. Same nomenclature you use talking about vesicular trafficking. In the nerve, we said if [vesicles] go from the cell body to the axon, you go anterograde. Vesicles that go from the end of the axons back to the nucleus/cell body are retrograde.

Trans Golgi Network

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. When you get to the TGN, you're packaged into vesicles. Put aside the clathrin-coated vesicles going to the lysosome. Two other types of vesicles: 1) Forms, in a constitutive fashion, goes to the plasma membrane and dumps the components outside of the cell. This is an unregulated membrane fusion. What's this mean? The clotting enzymes that are policing everything are made in the liver. There is a certain concentration in the blood at all times. Why? Because hepatocytes are making those enzymes and dumping them out there. Or ECM. You have to always replenish it. All cells are constitutively secreting matrix proteins. That is an unregulated constitutive release mechanism. You can replenish plasma membrane, proteins, and you can dump things outside of cell. In contrast, there are proteins that are packaged into vesicles and sit there until they are needed within the cytoplasm. Good example is insulin. Insulin is in granules already in the pancreas - sitting in beta cells. When you eat, and your sugar levels go up, you induce a signal to release insulin for metabolism. That is called a regulated secretory pathway. An example is mast cells (A mast cell (also known as a mastocyte or a labrocyte) is a type of white blood cell. Specifically, it is a type of granulocyte derived from the myeloid stem cell that is a part of the immune and neuroimmune systems and contains many granules rich in histamine and heparin.) The mast cells (on right). In the granules are histamine and heparin. When they are needed, you get a degranulation. All these vesicles fuse with the plasma membrane. That is a regulated release mechanism. This is an EM of degranulation. When we talk about vesicular release, that is the same as merocrine.

Functions of glycosylation

There are multiple. Adhesion. We've talked about selectins, sugar moieties on the outside of membranes, receptors on certain cells that will target those cells to sugar moieties. 1. Recognition [I believe this is adhesion] 2. Solubility 3. Protection: [Dr. Lerea] always thinks of the northwest. You have forests, you want to protect individuals going in for logging. You basically have a chain saw but if you have a lot of branches on a tree, sometimes it is tough to get to that trunk. The sugar moieties are like the branches. They are protecting the amino acid polypeptide backbone from proteases.

nucleus, ER, golgi

You have to go to EM to appreciate the Golgi. You can see, to orient you, the nucleus [N]. The membrane [pointed to by the arrows] is of the rough ER. You know because you see the membrane stacks studded with the ribosomes. You can also see the golgi apparatus, the cis face and the trans face. On stain, clear often means an extensive golgi apparatus because the golgi doesn't pick up any stain.

endoplasmic reticulum

a network of membranous tubules within the cytoplasm of a eukaryotic cell, continuous with the nuclear membrane. It usually has ribosomes attached and is involved in protein and lipid synthesis. You know when a protein is made at the level of the rough ER, you have chaperones that first keep it in an unfolded state, and then help fold it to the correct state. Certain reactions go on. For example, disulfide isomerases may form disulfide bridges if the protein is folded in the correct conformation. You can also get N-linked glycosylations, and so forth. All these processes are needed to move a protein out of the ER and into the Golgi and so forth. If something is not correct, it may just stop there and do damage.

Proteasomes

a protein complex in cells containing proteases; it breaks down proteins that have been tagged by ubiquitin. Proteins for degradation are being brought from outside. What is the major degradation pathway in cytoplasm? Proteasomes. Poly ubiquitination goes with the proteasome. Mono ubiquitination goes with tagging receptors to have those cytoplasm tails to be degraded further.